Journal: iScience

Article Title: A combinatorial CAAR-T cell strategy to eliminate factor VIII inhibitors in preclinical models of hemophilia A

doi: 10.1016/j.isci.2026.114924

Figure Lengend Snippet: Construction of CAAR-T cells targeting FVIII inhibitors and their functional characterization (A) Schematic diagram of FVIII proteolytic fragments. The domain structure of the FVIII protein after physiological cleavage is shown, illustrating its division into a heavy chain (A1-A2) and light chain (A3-C1-C2). (B) Design of chimeric autoantibody receptor constructs. Two CAAR constructs were generated using the A2 or C1 domains of FVIII as target antigens. Each CAAR consists of the FVIII-derived domain fused to CD28 transmembrane and co-stimulatory regions, CD3ζ signaling domain, and a P2A self-cleaving peptide linked to GFP for expression tracking. (C) Flow cytometric analysis of CAAR expression on human T cells. Top, GFP reporter was used to assess transduction efficiency of A2-CAAR and C1-CAAR constructs, with both showing high GFP + proportions compared to control T cells. Bottom, HA-tag detection was used to confirm surface expression of CAAR constructs; the HA-tag was inserted between the A2 or C1 extracellular domain and the CD28 transmembrane region within the CAAR plasmid. (D) Characterization of engineered K562 target cells. BOIIB2- and RHD5-expressing K562 cells were sorted by flow cytometry based on strong mCherry fluorescence, achieving >97% purity in both lines. (E) Cytotoxic activity of CAAR-T cells against antigen-expressing target cells. Cytotoxicity assays were performed using LDH release to evaluate the lytic activity of A2-CAAR T and C1-CAAR T cells at various effector-to-target (E:T) ratios (0.3:1, 1:1, and 3:1). A2-CAAR T cells were tested against BOIIB2 targets, and C1-CAAR T cells against RHD5 targets. UTD were used as controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 3 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (F) IFN-γ secretion by CAAR-T cells upon target recognition. ELISA quantification of IFN-γ released by CAAR-T cells co-cultured with their respective antigen-expressing targets at an E:T ratio of 3:1. Both A2-CAAR T and C1-CAAR T cells showed significantly increased cytokine release compared to UTD controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 3 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Degranulation activity of CAAR-T cells assessed by CD107α expression. Flow cytometry analysis of CD107α surface mobilization in CAAR-T cells after 4 h co-culture with target cells at an E:T ratio of 3:1. Quantification and mean fluorescence intensity (MFI) demonstrates elevated degranulation in both A2-CAAR and C1-CAAR groups compared to UTD controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 2 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CAAR-T cells and target cells were co-cultured in 96-well plates at various effector-to-target ratios for 18 h. Following incubation, the cytotoxicity was evaluated using a commercial LDH release assay kit (DOJINDO, CK12) according to the manufacturer’s protocol.

Techniques: Functional Assay, Construct, Generated, Derivative Assay, Expressing, Transduction, Control, Plasmid Preparation, Flow Cytometry, Fluorescence, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay